NO319146B1 - Microbiological process for the preparation of heteroaromatic carboxylic acids and picolinic acid or their physiologically tolerable salts by means of microorganisms from the genus Alcaligenes - Google Patents

Abstract

Prepn. of heteroaromatic carboxylic acids, or their salts, of formula (I) or (II) from heteroaromatic nitriles of formula (III) or (IV) by use of microorganisms of the genus Alcaligenes cultured in the presence of a di- or tri-carboxylic acid or a carbohydrate. R<1>, R<2> = H or Hal., and X = N or C.

Description

The invention relates to a new microbiological method for the production of heteroaromatic carboxylic acids or their physiologically tolerable salts with the general formulas:

wherein R<1>, R<2> are the same or different and mean a hydrogen or halogen atom, and X a nitrogen atom or -CH-.

Heteroaromatic carboxylic acids such as e.g. 6-hydroxypicolinic acid, are important intermediates for the production of pharmaceuticals such as for the production of 2-oxypyrimidine (Berichte der Deutschen Chemischen Gesellschaft 1912,45, p.2456-2467) or for the production of herbicides (EP-A 0 447 004).

In general, it is known that microorganisms containing nitrile hydratases and amidases or nitrilases convert nitriles into the corresponding acids.

For example, EP A 0 187 680 describes a microbiological method for the production of organic acids such as nicotinic acid, using microorganisms of the genus Corynebacterium, Nocardia, Bacillus, Bacteridium, Micrococcus and Brevibacterium. This reaction is carried out obligatorily in the presence of light energy. From EP-A 0 444 640, a microbiological method for the production of organic acids such as e.g. nicotinic acid, using microorganisms of the genus Rodococcus. This turnover is obligatorily carried out in the presence of a lactam.

Furthermore, it is known that microorganisms of the species Rodococcus rhodochrous J1 convert e.g. 2-cyanopyrazine to pyrazine carboxylic acid (Kobayashi et al., J. of Antibiotics, Vol. 43, No. 10, 1990, pp. 1316-1320).

However, these microorganisms cannot convert 2-cyanopyridine to picolinic acid (Mathew et al., Appl. Environmental Microbiology, Vol. 54, No. 4, 1988, pp. 1030-1032).

It is also known that 2-cyanopyridine-utilizing microorganisms of the genus Alcaligenes convert 2-cyanopyridine to 6-hydroxypicolinic acid (EP-A 0 504 818). With this method, it is a disadvantage that the 6-hydroxypicolinic acid is only formed in moderate yield.

The task of the present invention was to provide an economical microbiological method for the production of heteroaromatic carboxylic acids or their physiologically tolerable salts, such as pyrazine carboxylic acid, picolinic acid or chromium picolinate, by means of microorganisms of the genus Alcaligenes, during which the formed carboxylic acids or their physiologically tolerable salts. is formed in good yield.

This task was solved by the method according to claim 1.

According to the invention, the method is carried out by reacting a heteroaromatic nitrile with the general formulas as substrate

wherein X, R<1> and R<2> have the aforementioned meaning, by means of 2-cyanopyridine-metabolizing microorganisms of the genus Alcaligenes, which before the biotransformation were cultivated in the presence of a dicarboxylic acid, tricarboxylic acid or a carbohydrate, to heteroaromatic carboxylic acids of formula I or II. Heteroaromatic carboxylic acids are then optionally transferred into physiologically tolerable salts. Physiologically tolerable salts of these carboxylic acids are understood in the following to be respectively chromium, calcium or ammonium salts.

Before the actual biotransformation, microorganisms of the genus Alcaligenes used for the method are cultivated in the usual way, and their active enzymes are appropriately induced with 2-cyanopyridine.

For cultivation and induction, 2-cyanopyridine can be used in a concentration of 0.01 to 20% by weight, preferably in a concentration of 0.1 to 1% by weight.

In the following, a dicarboxylic acid is understood to mean fumaric acid, succinic acid, malic acid, glutaric acid, malonic acid, respectively their salts and derivatives as well as esters.

In the following, a tricarboxylic acid is understood to mean citric acid, isocitric acid, respectively their salts and derivatives such as esters. Fumarate, malate, malonate, oxaloacetate, citrate, aconitate, isocitrate, 2-oxoglutarate, succinate or succinyl-CoA can be used as salts and derivatives of these dicarboxylic acids and tricarboxylic acids. Fumarate, malonate or succinate are preferably used.

In the following, monosaccharides such as glucose, disaccharides such as sucrose, trehalose or maltose, trisaccharides such as raffinose, sugar alcohols such as glycerol are used as carbohydrates. Glycerol is preferably used as carbohydrate.

Appropriately, the dicarboxylic acid, the tricarboxylic acid, respectively the carbohydrate is used in a concentration of from 0.1 to 20% by weight, preferably in a concentration of 0.5 to 5% by weight.

As culture medium, the media that are common in the field can be used, such as e.g. the mineral salt medium according to Kulla et al. (Arch. Mrcrobiol., 135, 1-7, 1983), low molar phosphate buffers or the one according to Table 1. The one described in Table 1 is preferably used.

After the cultivation phase, or before the actual substrate addition, the microorganisms are harvested either by means of a separation process or the substrate is added directly to the microorganisms.

The substrates used for the biotransformation, the heteroaromatic nitriles of formula II and IV, which e.g. 2-cyanopyridine, are compounds in commerce.

In the general formulas 1-IV, X means a nitrogen atom or -CH-, preferably -CH-. The rest of R<1> and R2 are the same or different and mean hydrogen or halogen such as fluorine, chlorine, bromine or iodine.

Possible substrates are consequently 2-cyanopyridine, 6-chloro-2-cyanopyridine, 5,6-dichloro-2-cyanopyridine, 2-cyanopyrazine, 6-chloro-2-cyanopyrazine, 5-bromo-6-chloro-2-cyanopyrazine. Appropriately, 2-cyanopyridine, 2-cyanopyrazine or 6-chloro-2-cyanopyridine are used as substrates.

The substrate can be added for the biotransformation once or continuously. Appropriately, the substrate addition takes place so that the substrate concentration in the medium does not exceed 20% by weight, preferably does not exceed 10% by weight.

The biotransformation which is normally carried out with resting cells is conveniently carried out with the known 2-cyanopyridine-metabolizing microorganisms of the species Alcaligenes faecalis with the designation DSM 6335 known from EP-A 0 504 818 as well as with the functionally equivalent variants and mutants. This microorganism was deposited on January 3, 1991 at der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124. Braunschweig, according to the Budapest Convention.

"Functionally equivalent variants and mutants" means microorganisms that essentially have the same properties and functions as the original microorganisms. Such variants and mutants can be formed randomly, e.g. by means of UV irradiation.

For biotransformation, the same media can be used as for cultivating the microorganisms.

The biotransformation can also take place in the presence or absence of the previously described dicarboxylic acids, tricarboxylic acids, respectively carbohydrates.

The pH value is suitably in a range of 4 to 10, preferably in a range of 5 to 9. The biotransformation can be carried out at a temperature of 10 to 50°C, preferably at a temperature of 20 to 40°C.

After a normal reaction time of 6 to 100 hours, the corresponding carboxylic acids with formula I or II can then be obtained through normal work-up methods, such as e.g. by acidification. The carboxylic acids can also be isolated in the form of salts such as ammonium or chromium salt.

If produced as heteroaromatic carboxylic acids in position 6 hydroxylated heteroaromatic carboxylic acids (general formula II), the biotransformation is conveniently carried out under aerobic conditions. However, if a non-hydroxylated heteroaromatic carboxylic acid such as e.g. picolinic acid, the biotransformation is suitably carried out under anaerobic conditions.

Examples

Example 1

Preparation of 6-hydroxypicolinic acid

The following conditions were chosen for the production of 6-hydroxypicolinic acid with the strain Alcaligenes faecalis DSM 6335. A 7.5 L fermenter with a 5 L working volume was used. Alcaligenes faecalis DSM 6335 was grown in a mineral salt medium (Table I) with sodium fumarate as sole carbon and energy source, and 2-cyanopyridine as inducer at 30°C, 600 rpm, and a pH value of 7.0. The ventilation rate was below approx. 3 l/min. The addition of the sodium fumarate then took place p02-controlled at a p02 of >30%. A 20% stock solution of sodium fumarate mixed with 0.5% 2-cyanopyridine was used. The cells were grown to an optical density, measured at 650 nm (ODeso), of 16 within 23 hours before the biotransformation was started. For the growth phase, approx. 160 g of sodium fumarate in the form of a 20% solution (approx. 800 ml).

During the aerobic biotransformation of 2-cyanopyridine to 6-hydroxypicolinic acid, no carbon and energy source was added. The biotransformation took place with resting cells.

The addition of the 2-cyanopyridine took place limitingly by means of a pump. The pumping speed was then monitored "on line" using HPLC. The concentration of the intermediate picolinic acid, whose rate of formation is approx. 2.5 times higher than the conversion rate of picolinic acid to 6-hydroxypicolinic acid (10 g/l/h to 4g/l/h) was limited to values less than 2 g/l, as the conversion of picolinic acid to 6-hydroxypicolinic acid was otherwise inhibited.

As 2-cyanopyridine is a solid at room temperature, it was necessary to heat the starting container containing 2-cyanopyridine to 50°C so that 2-cyanopyridine could be added in liquid form.

With this method it was possible to produce 75 g/l of 6-hydroxypicolinic acid within 31 hours.

The intermediate picolinic acid could no longer be detected at the end of the biotransformation.

For the isolation of 6-hydroxypicolinic acid, the cells were separated by filtration. The cell-free solution was then heated to 60°C and acidified with concentrated hydrochloric acid to a pH of 2-2.5. At this pH, the 6-hydroxypicolinic acid precipitated from the solution.

It was then slowly cooled to 4°C with stirring, filtered, the residue washed with mineral-free water and dried (100 mbar, 55°C).

In the mother liquor, approx. 2g/l 6-hydroxypicolinic acid. The yield was 87% calculated on the starting material 2-cyanopyridine.

Example 2

Production of picolinic acid

Cultivation of the biomass took place as described in Example 1. The formation of picolinic acid took place under strictly anaerobic conditions.

For the biotransformation, a 500 ml glass bottle with a rubber septum was used, filled with 400 ml biomass of ODe5o= 20. It was incubated at 30°C. Before starting the biotransformation, the batch was made anaerobic with pure nitrogen. For this, the batch was gassed for 30 min. through cannulae while stirring with nitrogen (50 mbar excess pressure) to expel the oxygen quantitatively. In order to exclude oxygen entry during the biotransformation or by the addition of 2-cyanopyridine, gassing was maintained during the biotransformation (approx. 10 mbar overpressure).

The addition of 2-cyanopyridine took place in 12 steps of 10 g/l each time after a period of 1 hour. However, the addition can also take place continuously. Using HPLC, it was investigated whether 2-cyanopyridine had been completely converted into picolinic acid before adding a further portion. During the biotransformation, no formation of picolinic acid amide could be detected.

With this method it was possible to produce approx. 150g/l picolinic acid within 26 hours. 6-Hydroxypicolinic acid was not formed there.

For the isolation, the cell-free picolinic acid solution was precipitated with CaCl2/H2SO4. For this, the cell-free picolinic acid solution from Ekempel 2 was diluted 3 times and mixed with stirring with 0.5 equivalents of CaCI2 per equivalent of picolinic acid after the cell-free fermentation solution had been preheated to 90°C. The formed calcium-picolinic acid complex immediately fell out. The complex formed was cooled to 4°C with stirring, filtered through a glass filter (porosity 3), and washed with mineral-free water.

The filter cake was slurried in mineral-free water and acidified with concentrated sulfuric acid to a pH of 2.5. Underneath, the picolinic acid was dissolved from the complex and at the same time insoluble calcium sulfate was formed. As the free picolinic acid is very water-soluble, calcium sulphate could be separated by filtration. The picolinic acid solution was evaporated to dryness and analyzed. The foot yield was approx. 70% with a purity of 86% according to titration. The water content was 0.7% measured with the Karl-Fischer method.

Example 3

Preparation of chromium(III) picolinate

To an ammonium picolinate solution (271.4 g; 0.325 mol; 16.8%), placed in a 500 ml flask, at 73°C aqueous chromium trichloride hexahydrate solution (23.95 g, 0.09 mol chromium in 63 ml water) over a period of 3.5 hours. The violet solution obtained was stirred for another hour and then cooled slowly to 3°C. After precipitation of the red solid formed, the upper blue phase was decanted. The solid was slurried with 100 ml of water for 30 minutes and then again decanted from. After a second slurry with 50 ml of water (30 min.), the solid was filtered off and dried at 50°C under vacuum. 33.64 g of dark red crystals were obtained (90% yield).

Example 4

Cultivation of Alcaligenes faecalis DSM 6335 with different carbon sources

For cultivation of Alcaligenes faecalis (DSM 6335) 300 ml Erlenmeyer flasks with 100 ml A + N Medium (Table 1 without fumaric acid disodium juice) were used. The medium was additionally mixed with 2g/l 2-cyanopyridine and 10g/l of the following carbon sources:

Fumaric acid-dinatirum salt

Glycerol

Malonic acid disodium salt

Succinic acid disodium salt

Incubation took place on a shaking machine at 30°C. After 16 hours of growth, the cells were centrifuged off, and the fresh A + N medium (without carbon source) containing 10g/l 2-cyanopyridine resuspended. The optical density of the cell suspension measured at 650 nm (ODeso) was 10. Then the cell suspension (total volume 10-20 ml) was re-incubated at 30°C. The formation of 6-hydroxypicolinic acid was followed spectrophotometrically by measuring the absorbance of the cell-free solution at 308 nm. The following average productivities were determined for the formation of 6-hydroxypicolinic acid:

Example 5

Preparation of 6-hydroxypyrazine carboxylic acid

The cultivation of Alcaligenes faecalis (DSM 6335) took place as in Example 4 with fumaric acid as carbon source. The washed cells were resuspended in A+N medium containing 10 g/l 2-cyanopyrazine (OD650=10) and incubated at 30°C. The formation of 6-hydroxypyrazine carboxylic acid was followed spectrophotometrically by measuring the absorbance of the cell-free solution at 320 nm. The reduction of the concentration of 2-cyanopyrazine (substrate) could be determined by measuring the absorbance at 270 nm. After 7 hours, the introduced amount of 2-cyanopyrazine had been converted to 6-hydroxypyrazine carboxylic acid.

Example 6

Preparation of 6-chloropicolinic acid and pyrazine carboxylic acid

Cultivation of Alcaligenes faecalis (DSM 6335) took place as in Example 4 with fumaric acid as carbon source. The washed cells were resuspended in A+N medium in shards of glass (ODeso - 10) that could be closed with rubber stoppers, and gassed using cannulas with nitrogen to remove dissolved oxygen. The cell suspensions were then added with 2-cyanopyrazine or 6-chloro-2-cyanopyridine as substrate to a final concentration of 10g/L and incubated at 30°C. After 3 hours, the starting substances had been quantitatively converted to the corresponding acids [Detection by thin-layer chromatography; silica gel 60 with fluorescence indicator, eluent: chloroform 30 / ethanol 55 / NH4OH(25%) 10 / H20 5].

Claims (4)

1. Microbiological process for the production of heteroaromatic carboxylic acids or their physiologically tolerable salts with the general formulas: in which R<1>, R2 are the same or different, and mean a hydrogen or halogen atom and X a nitrogen atom or -CH-, characterized in that the substrate is reacted with heteroaromatic nitriles with the general formulas: wherein R<1>, R2 and X have the aforementioned meaning, by means of 2-cyanopyridine-metabolizing microorganisms of the genus Alcaligenes, which before the biotransformation are cultivated in the presence of a dicarboxylic acid, tricarboxylic acid or a carbohydrate, to the corresponding carboxylic acid, and optionally converts the latter into physiologically tolerable salts. 2. Microbiological method according to claim 1, characterized in that the biotransformation is carried out with microorganisms of the species Alcaligenes faecalis with the designation DSM 6335 and with their functionally equivalent variants and mutants. 3. Microbiological method according to claim 1 or 2, characterized in that the biotransformation is carried out at a pH of 4 to 10 and a temperature of 10 to 50°C. 4. Microbiological process for the production of picolinic acid and its physiologically tolerable salts, characterized in that 2-cyanopicolin is converted as a substrate by means of 2-cyanopyridine-metabolizing microorganisms of the genus Alcaligenes, which before the biotransformation were cultivated in the presence of a dicarboxylic acid, under anaerobic conditions, to picolinic acid, and optionally transfers the latter in the physiologically tolerable salt.